O. P. SrivastavaO. P. SrivastavaDepartment of Vision Science Department of Vision Science

University of Alabama at University of Alabama at BirminghamBirmingham

Physical Methods in Models of Cataract Disease

Function of the Lens:

Refraction

Lens Specific Structural Proteins Lens Specific Structural Proteins ((αα--, , ββ-- and and γγ--CrystallinsCrystallins))

αα--Crystallin:Crystallin:Two primary gene Two primary gene products (products (ααA and A and ααB, B, both 20 kDa), 800 kDa both 20 kDa), 800 kDa oligomer (oligomer (ααA:A:ααB, 3:1), B, 3:1), Chaperone activity. Chaperone activity.

ββ--Crystallin:Crystallin:Eight primary gene Eight primary gene products, 23products, 23--32 kDa 32 kDa (Acidic (Acidic ββA3/A1, A3/A1, ββA3 and A3 and ββA4, Basic A4, Basic ββB1, B1, ββB2, B2, ββB3 and B3 and ββB4), 50B4), 50--200 200 kDa oligomerkDa oligomer

γγ--Crystallin: Crystallin: Six primary Six primary gene products, 20 kDa gene products, 20 kDa ((γγA, A, γγB, B, γγC, C, γγD, D, γγE and E and γγF), monomer.F), monomer.

HMW: >1X106 D

α: 800 kDa

βL60 kDa

γ:20 kDa

βH: 200 kDa

Normal lens

Cataractous lenses

National Eye Institute (NIH)National Eye Institute (NIH)

Most cataracts are related to aging. Most cataracts are related to aging.

By age 80, more than half of all By age 80, more than half of all Americans either have a cataract or Americans either have a cataract or have had cataract surgery.have had cataract surgery.

Estimated Specific Prevalence Rates for Estimated Specific Prevalence Rates for CataractCataract

Source: National Eye InstituteSource: National Eye Institute

Female Male

Summary of Eye Disease Prevalence Data Source: National Eye Institute

Age, Cataract Advanced AMDIntermediate

AMD Glaucoma

Years Persons (%) Persons (%) Persons (%) Persons (%)

40-49 1,046,000 2.5% 20,000 0.1% 851,000 2.0% 290,000 0.7%

50-59 2,123,000 6.8% 113,000 0.4% 1,053,000 3.4% 318,000 1.0%

60-69 4,061,000 20.0% 147,000 0.7% 1,294,000 6.4% 369,000 1.8%

70-79 6,973,000 42.8% 388,000 2.4% 1,949,000 12.0% 530,000 3.9%

>80 6,272,000 68.3% 1,081,000 11.8% 2,164,000 23.6% 711,000 7.7%

Total 20,475,000 17.2% 1,749,000 1.5% 7,311,000 6.1% 2,218,000 1.9%

ICR/f Rat Model: Study Effects of ICR/f Rat Model: Study Effects of Botanicals on Mechanism of AgeBotanicals on Mechanism of Age--

Related Human CataractRelated Human Cataract

Cataract Disease in RatsSprague-Dawley Rat

ICR/f Rat

A colony of ICR/f rats has been established

Development of Lens Opacity in ICR/f Rats

30 Days No Opacity

60 Days A diffused opacity at posterior subcapsular area ( around

suture)

90 Days Dense opacity at the posterior polar

region, rapidly extends to whole cortical region, then to posterior

nuclear region and finally to whole nuclear region

120 days Opacity of whole lens

Time-Line

Grape Seed Extract Slows Onset of Grape Seed Extract Slows Onset of Cataract Disease in ICR/f RatsCataract Disease in ICR/f Rats

ICR/f rats imported from Meijo University in Japan to create a breeding colony at UAB

Inbred strain derived from the original ICR rats

Cataract formation significantly slowed by 0.2% grape seed extract in the diet (Yamakoshi et al., 2002)

New experiments will evaluate a dose-response curve for PACNs (0.1-5%)

Control at <30 days

GSE diet; 27 days later

Control diet; 27 days later

ICR/f Rats (Cataract inherited through an autosomal recessive gene)

10X higher Ca+2

Activation of m-calpain and

transglutamase

GSH and water soluble proteins

decrease, oxidation of Met

Increasein HMW proteins

Increase in C-terminally

truncated αA and αB-crystallin

Racemization and isomerization of Asp-151 of αA-crystallin

Increased phosphorylation of Ser

of C-terminally truncated αA and αB-

crystallins

Cross-linking of αA-

crystallin

(Aggregation and Cross-Linking)

γ, βB1 and βA3-

crystallins decreased

Increase in lipid

peroxide in serum

(Oxidative Insult) (Racemization)

(Increased Phosphorylation)(Proteolysis)

What is Molecular Mechanism of What is Molecular Mechanism of Cataract Development in ICR/f Cataract Development in ICR/f

Rats?Rats?

How Do AgeHow Do Age--Related Cataracts Related Cataracts Develop?Develop?

Clumps of protein accumulate in lens Clumps of protein accumulate in lens and become insoluble causing opacity.and become insoluble causing opacity.

The clear lens slowly changes to a The clear lens slowly changes to a yellowish/brownish color, adding a yellowish/brownish color, adding a brownish tint to vision. brownish tint to vision.

Source: National Eye Institute

Lens α-, β- and γ-Crystallins [Water Soluble]

Conformation Changes

Aggregation (Hydrophobic interactions)[Water Soluble-HMW- and Water Insoluble Proteins]

Covalent Cross-Linking(Disulfide and non-disulfide types)

[Water Soluble and Water Insoluble]

Opacity [Water Insoluble]

Post-translational modifications during

Aging

HYPOTHESIS

Conformational Changes Leading to Conformational Changes Leading to Aggregation and CrossAggregation and Cross--LinkingLinking

Racemization

Increasedphosphorylation

Proteolysis

Oxidative insult

Conformational changes

Aggregation and

Cross-linking

Conformational Changes in Conformational Changes in Proteins and Protein AggregatesProteins and Protein AggregatesTwoTwo--Dimensional gel Electrophoresis Dimensional gel Electrophoresis and Mass Spectrometric Analysesand Mass Spectrometric AnalysesMolecular weights of Aggregates and Molecular weights of Aggregates and Identification of their components Identification of their components (Dynamic Light(Dynamic Light--Scattering and BlueScattering and Blue--native Gel Electrophoresis) native Gel Electrophoresis) Secondary and Tertiary Structures of Secondary and Tertiary Structures of Modified Proteins Modified Proteins (CD Spectroscopy)(CD Spectroscopy)Structural Changes in Modified Structural Changes in Modified Proteins Proteins (Determination of (Determination of hydrophobicity and Trp fluorescence)hydrophobicity and Trp fluorescence)

Fractionation of Human Lens Fractionation of Human Lens ProteinsProteins

Lens HomogenateLens HomogenateCentrifuge

Water Soluble Protein Fraction

Water Insoluble Protein Fraction

Urea Soluble Fraction

Urea Insoluble Fraction

Centrifuge Urea Solubilization

HMW Protein Fraction

(>1 X106 D)

Changes with aging and cataract

#21 Old_highSoy

β-B2-crystallinsβ-A3-crystallins

α-A crystallins α-B crystallins20

10

253750

250

15

100150

75

#89 Young_Control

#57 Old_control

β-B2-crystallinsβ-A3-crystallins

α-A crystallinsα-B crystallins

β-B2-crystallinsβ-A3-crystallins

α-A crystallins α-B crystallins

2DE-Profiles of Lens WS Proteins fromMonkeys Fed Soy

Molecular Mass of Protein Molecular Mass of Protein Aggregates and Identification of Aggregates and Identification of

their Components their Components

(Dynamic Light(Dynamic Light--Scattering Method and Scattering Method and BlueBlue-- native Gel Electrophoresis)native Gel Electrophoresis)

Determination of Molecular Weights and Determination of Molecular Weights and Hydrodynamic Radai of WSHydrodynamic Radai of WS--HMW ProteinsHMW Proteins

WS-Proteins

Size-exclusion HPLC using TSK G-5000 PWXL Column

Determine absolute molar mass and hydrodynamic radius

Monitor protein absorbance, light

scattering by QUELS (quasielastic light

scattering device)and refractive index

(DAWN HELEOS)

Time (Min)

A280

Protein Profile of WS-Proteins from a 60 Year-Old Human Donor

Light scattering A280

Refractive index

WS-HMW Proteins in Aging Human Lenses After Size-Exclusion Chromatography

Molecular Mass of HMW Proteins Molecular Mass of HMW Proteins from Human Lensesfrom Human Lenses

human lens (years)

0 20 40 60 80 100

Abs

olut

e m

olec

ular

mas

s

0

200

400

600

800

1000

1200

1400

1600

860 kDa 955 kDa

1196 kDa

1468 kDa

2D-Blue Native Gel Electrophoresis to analyze Protein Aggregates

Secondary and Tertiary Secondary and Tertiary Structures of Modified Proteins Structures of Modified Proteins

(CD Spectroscopy)(CD Spectroscopy)

Circular Dichroism Spectroscopy Circular Dichroism Spectroscopy is Particularly Good for:is Particularly Good for:

SSecondary and econdary and tertiary structuretertiary structuress of of proteinsproteinsComparing Comparing structures for different mutantsstructures for different mutants of of the same proteinthe same proteinStudying the Studying the conformational stability of a conformational stability of a protein under stress protein under stress ---- thermal stabilitythermal stability, , pHpH stability, and stability to denaturantsstability, and stability to denaturantsFor finding For finding solvent conditions that increase solvent conditions that increase the melting temperature and/or the the melting temperature and/or the reversibility of thermal unfoldingreversibility of thermal unfolding conditions conditions which generally enhance shelf life.which generally enhance shelf life.Determining whether Determining whether proteinprotein--protein protein interactionsinteractions alter the conformation of protein.alter the conformation of protein.

Determination of Protein Secondary Determination of Protein Secondary Structure by Circular DichroismStructure by Circular DichroismSecondary structure Secondary structure can be determined by can be determined by CD spectroscopy in CD spectroscopy in the "farthe "far--uv" spectral uv" spectral region (190region (190--250 nm). 250 nm). At these wavelengths At these wavelengths the chromophore is the chromophore is the peptide bond, and the peptide bond, and the signal arises the signal arises when it is located in a when it is located in a regular, folded regular, folded environment. environment.

Polylysine

Mutants of Human Lens Beta A3Mutants of Human Lens Beta A3--CrystallinCrystallin

CD Spectra of Mutant Proteins of CD Spectra of Mutant Proteins of ββA3A3--CrystallinCrystallin

Absorption Spectra of Phe, Trp and Tyr

295 nm

Intrinsic Trp Fluorescence Spectra of Native and Denatured WT-βA3 and its eight Truncated Mutant Proteins.

Intrinsic Trp Fluorescence Spectra:•Excitation at 295 nm and emission between 300-400 nm.

•Quenching, red or blue shift suggest change in microenvironment of Trp residues.

βA3-crystallin contains 9Trp residues; two are exposed (139, 153), four are buried (73, 99, 168, and 198) and three are partially buried (36, 96, 195).

Fluorescence Spectra of WT βA3 and its Eight Deletion Mutant Proteins After ANS Binding

ANS (8-amilino 1-naphthalenesulfate):

•A hydrophobic probe

•Binding is assessed by fluorescence spectra (Excitation at 390 nm and emission between 400 to 600 nm)

•Quenching; Reduced binding

•Red Shift: Increased exposure of hydrophobic patches.

•Blue Shift: Decreased exposure of hydrophobic patches.